In this proposal, funds are requested for a confocal imaging system to support and enhance the work of several NIH funded investigations involving a variety of dynamic cellular systems. Central to most of these studies is the use of a unique 3-dimensional (3D), collagen gel culture system. Since embryonic reality can be more closely duplicated using the collagen gel culture system, it has gained wide acceptance as useful model for studying the mechanisms of development. Examples of the cell biological questions currently being studied which would be enhanced significantly by the availability of confocal optics include: 1) fate of cell surface adhesion molecules after induced epithelial/mesenchymal transformation in endothelial cells. 2) changes in cytoskeletal F actin in relation to modulation of cell-cell and cell-substrate adhesion molecules during induction of cardiac mesenchyme. 3) comparison of cell surface vs cytosolic distribution of growth factors (FGF, TGFb) in embryonic cells. 4) binding and fate (e.g. endocytosis) of peptide growth factor-like proteins with known inductive potential. It has been suggested that confocal microscopy is the most significant advance in biological light microscopy in this decade (Shotton and White, TIBS 14:435, 1989). Although to some the previous statement is arguable, in our minds confocal optics appears to be the ideal solution to the imaging problems inherent to the 3D culture system. For example, it will allow for: (a) non-invasive optical sectioning of intact and living cultures and increase lateral resolution, (b) elimination of out-of-focus blur present in whole mounts of cultures stained with fluorescent labelled markers, (c) determination of the distribution of labeled markers within cells or tissues by quantitative volumetric imaging using reconstruction of serial sets of optical sections and (d) 4D imaging, i.e. the production of animated sequences of cell motility and antigen or ion redistribution by combining serial sectioning with time-lapse analysis. It is important to note that these advantages are not only limited to analysis of 3D culture systems, but are also applicable to studies using more conventional culture systems or tissue preparations greater than 10um. All major users are from the Department of Anatomy and Cellular Biology at the Medical College of Wisconsin. All have Federal funding, nine from NIH and one from NASA. Of the 2.2 million in extramural funding generated by the department, 50-69% is related to work with the collagen gel culture model. This will be the only confocal system in the medical complex.